Contraction of vascular smooth muscle in the walls of blood vessels can decrease blood flow to a tissue or cause an elevation of pressure. Determining how contraction of vascular smooth muscle is regulated is clearly important to our understanding of the cardiovascular system. Biochemical studies on myosin isolated from smooth muscle have shown that one of the small subunits (light chains) can be phosphorylated by a specific enzyme, light chain kinase, found in smooth muscle. Based on these studies, myosin light chain phosphorylation has been proposed as the only determinant of whether smooth muscle contracts or relaxes. This hypothesis arose from studies of the ATPase rate of isolated proteins, but little is known about whether phosphorylation takes place in smooth muscle with intact cells while retaining the most characteristic feature of muscle, the ability to develop force and shorten. The objective of the proposed research is to determine the role, if any, of light chain phosphorylation in regulating the contraction of intact vascular smooth muscle, and the mechanisms involved. A sensitive method has been developed to quantitate the extent of light chain phosphorylation in small muscle strips using isoelectric focusing to detect the change in the isoelectric point due to phosphorylation. Smooth muscle strips will be prepared from the pig carotid artery, and their contractile responses to graded stimulation by different means will be quantified through length-tension and force velocity curves. These strips will be frozen at different levels of activation and at different times after stimulation and analyzed for light chain phosphorylation. Correlations, if any, between light chain phosphorylation and force, velocity or both should provide valuable inferences on how phosphorylation is involved in regulating contractile activity. Critical tests of the hypothesis that light chain phosphorylation is the only determinant of contractile activity will be made in this more physiological context by (1) studying the time course of phosphorylation compared to force development, and (2) determining if there are means of stimulation where phosphorylation will not be correlated with force. The experiments should provide valuable insights into the regulatory mechanisms of vascular smooth muscle contraction.